Method for prolonging the over survival of a cancer patient

ABSTRACT

The present invention relates to a method for treating a cancer with the combination of a topoisomerase II inhibitor (TOP2 inhibitor) and a histone deacetylation inhibitor (HDACi), provides synergistic cancer cell-killing effects, and prolonged overall survival, especially when the expression of checkpoint kinase 1 (CHK-1) is low.

RELATED APPLICATION

This application is a Divisional application of U.S. application Ser.No. 15/095,508 filed Apr. 11, 2016, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No.62/145,305 filed Apr. 9, 2015, the content of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to a method for treating acancer, particularly, the present invention relates to a method fortreating a cancer with a combination of an anti-cancer drug and ahistone deacetylation inhibitor (HDACi) after checking the expression ofcheckpoint kinase 1 (CHK-1).

BACKGROUND OF THE INVENTION

Hepatocelluar carcinoma (HCC), of which the prevalence in Asia Pacificarea including Taiwan is high and is an important cause of death(El-Serag H B, Rudolph K L. Hepatocellular carcinoma: epidemiology andmolecular carcinogenesis. Gastroenterology 2007; 132: 2557-2576).Surgical resection remains the way most promisingly leading to long-termsurvival; however, only 20% or less of the patients with HCC couldundertake surgical resection (Hung H. Treatment modalities forhepatocellular carcinoma. Curr Cancer Drug Targets 2005; 5: 131-138.).There is still an unmet need for systemic therapies for advanced HCC(Lopez P M, Villanueva A, Llovet J M. Systematic review: evidence basedmanagement of hepatocellular carcinoma—an updated analysis of randomizedcontrolled trials. Aliment Pharmacol Ther 2006; 23: 1535-1547) evenafter sorafenib was proven to improve overall survival (OS) in patientswith advanced HCC (Llovet J M, Ricci S, Mazzaferro V, et al. Sorafenibin advanced hepatocellular carcinoma. N Engl J Med 2008; 359: 378-390).Sorafenib significantly prolonged OS of patients with advanced HCC;however, the increment was small especially for patients in Asia Pacificarea where around 70% of HCC are HBV associated, with median OS of 6.5months in the sorafenib arm compared with 4.2 months in the placebo arm(Cheng A L, Kang Y K, Chen Z, Tsao C J, Qin S, Kim J S, Luo R, Feng J,Ye S, Yang T S, Xu J, Sun Y, Liang H, Liu J, Wang J, Tak W Y, Pan H,Burock K, Zou J, Voliotis D, Guan Z. Efficacy and safety of sorafenib inpatients in the Asia-Pacific region with advanced hepatocellularcarcinoma: a phase III randomised, double-blind, placebo-controlledtrial. Lancet Oncol. 2009 January; 10(1):25-34).

There is still a need for new systemic therapies for a cancer withsynergistic efficacies.

SUMMARY OF THE INVENTION

This invention is based on the unexpected finding that the combinationof a topoisomerase II inhibitor (TOP2 inhibitor), such as doxorubicin orhydrazaline, and a histone deacetylation inhibitor (HDACi), such assuberoylanilide hydroxamic acid (SAHA) or valproic acid, providessynergistic cancer cell-killing effects, and prolonged overall survival.

Furthermore, it is also found in the invention that checkpoint kinase 1(CHK-1) is potentially a biomarker of synergistic cytotoxicity of HDACiin combination with DSB-inducing chemotherapeutics, such as atopoisomerase II inhibitor (TOP2 inhibitor), when the expression ofCHK-1 is low in the subject.

Accordingly, in one aspect, the present invention provides method fortreating a cancer and prolonging the overall survival of a subjectsuffering a cancer comprising:

providing a cancer cell sample of the subject, and determining theexpression of CHK-1 of the sample, wherein the attenuation of activatedCHK-1 used as a biomarker;

administering the subject, if the attenuation of activated CHK-1 isfound in the sample, with a combination of a a topoisomerase IIinhibitor (TOP2 inhibitor) and a Histone deacetylation inhibitor (HDACi)at the ratio to provide synergistic cancer cell-killing effects andprolonged overall survival.

In one example of the invention, the HDACi is suberoylanilide hydroxamicacid (SAHA) or valproic acid (VA).

In one example of the invention, the TOP2 inhibitor is doxorubicin(Doxo) or hydrazaline (HZ).

In another aspect, the invention provides a pharmaceutical compositioncomprising a combination of valproic acid (VA) and hydrazaline (HZ) atthe ratio to provide synergistic cancer cell-killing effects andprolonged overall survival.

In one example of the invention, the cancer is hepatocellular carcinoma(HCC).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawing. In the drawings:

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawing. In the drawings:

FIG. 1 shows that progression free survivals of patients treated with VAand HZ modulated GCGG (A), DoxDTIC (B), and the 2-step treatments (C),and overall survival of the patients with advanced HCC following the VAand HZ modulated 2-step chemotherapy (D).

FIG. 2(A) provides the results of the isobolograms analysis for SAHA incombination with Doxo in CTV-1 and Molt-3 T-ALL cells (wherein the dataare presented as means±SE for at least three independent experiments).

FIG. 2(B) provides the results of the isobolograms analysis for VPA incombination with Doxo in J5, PLC and Hep3B HCC cells (wherein the dataare presented as means±SE for at least three independent experiments).

FIG. 3 shows that SAHA sensitized CTV-1 cells to doxo-induced apoptosis,wherein the apoptosis was found with the increased M2 fractions by TUNELassay (A), and the increased cleavage of caspase 8 and/or 9 by westernblot (B).

FIG. 4 A provides the cell cycle distributions of CTV-1 after 12-hourtreatment with doxorubicin (Doxo), suberoylanilide hydroxamic acid(SAHA) at IC50 or combinations, analyzed with BrdU assay (* denotesp<0.05, comparing cell percentages between after Doxo and Doxo plus SAHAtreatment).

FIG. 5(A) provides the representive western blots for CHK-1, p-CHK-1,CHK-2 and p-CHK-2 in J5, PLC, Hep3B HCC cells treated with vehicles only(Doxo:-, and VPA: -), VPA, Doxo, or both VPA and Doxo, wherein the lowintrinsic CHK-1 expression (Doxo:-, and VPA: -) led to relatively lowp-CHK-1 expression in Hep3B cells comparing to in J5 and PLC cells.

FIG. 5(B) shows the result of the CHK-1 expression standardized byloading quantities, indicating that the expression of CHK-1 wassignificantly low in Hep3B cells without exposure to VPA or Doxo orexposure to both Doxo and VPA.

FIG. 5 (C) shows another independent test for CHK-1 expression,indicating the similar results.

DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a sample” includes a plurality of such samplesand equivalents thereof known to those skilled in the art.

The drugs targeting topoisomerase II (TOP2) have been in clinical usefor many years, such as etoposide, doxorubicin and mitoxantrone and arefound to reduce the toxicity when used in combination of otheranticancer agents, including chemotherapeutic agents.

Histone deacetylation inhibitors (HDACi) are involved in acetylation anddegradation of Sae2 (orthologue of human CtIP) required for DNA doublestrand break repair (Robert T, Vanoli F, Chiolo I, Shubassi G, BernsteinK A, Rothstein R, Botrugno O A, Parazzoli D, Oldani A, Minucci S, FoianiM. HDACs link the DNA damage response, processing of double-strandbreaks and autophagy. Nature. 2011; 471(7336):74-9). HDACis may thusabort DNA repair in tumor cells after chemotherapy and promote efficacyof chemotherapy which may lead to double strand DNA break. Examples ofHDACis include but are not limited to suberoylanilide hydroxamic acid(SAHA), Valproic acid (VA) and hydrazaline (HZ). Valproic acid (VA) andhydrazaline (HZ) can function as HDACis and may after chemotherapy abortDNA repair in tumor cells and thus promote efficacy of chemotherapy.

It is unexpectedly found in the invention that attenuation of CHK-1expression in T-ALL cells and hepatocellular cells was associated withproapoptosis and hampering of DNA repair when double strand DNA breakswere chemotherapy-induced. It was evidenced in the invention that thesynergistic cell-killing effects of the combination of doxorubicin (aTOP2 inhibitor) and suberoylanilide hydroxamic acid (SAHA) (an HDACi) ona CTV-lymphoblastic cell line of which phosphorylation of CHK-1 werenegatively affected by SAHA. However, in the SAHA-treated T ALL cells,the repair of doxorubicin-induced double strand DNA break (DSB) wasinitiated with increased H2AX. However, although SAHA increased H2AX andacetyl H2AX, DSB repair was aborted by decreasing G2/M arrest of cellsand hampering the nuclear entry of CtIP, a DNA repair protein. SAHAeventually synergistically increased doxorubicin-induced apoptosis withthe induction of cleaved caspases 8 and 9. In addition, valproic acidshowed synergistic effects with doxorubicin in inducing cell death ofHep3 and PLC hepatocellular carcinoma (HCC) cells. HDACi thussynergistically impaired the topoisomerase II-induced DSB repair andinduced apoptosis by eliciting DNA repair with H2AX; however, abortedthe DNA repair and induced apoptosis by hampering the nuclear entry ofCtIP. Attenuation of activated CHK-1 may be potentially a biomarkers ofsynergistic cytotoxicities of HDACi and DSB-inducing chemotherapeutics.

Accordingly, the invention provides A method for treating a cancer andprolonging the overall survival of a subject suffering a cancercomprising:

providing a cancer cell sample of the subject, and determining theexpression of (CHK-1) of the sample, wherein the attenuation ofactivated CHK-1 used as a biomarker;

providing a cancer cell sample of the subject, and determining theexpression of checkpoint kinase 1 (CHK-1) of the sample, wherein theattenuation of activated CHK-1 is used as a biomarker; and

administering the subject, if the attenuation of activated CHK-1 isfound in the sample, with a combination of a a topoisomerase IIinhibitor (TOP2 inhibitor) and a Histone deacetylation inhibitor (HDACi)at the ratio to provide synergistic cancer cell-killing effects andprolonged overall survival.

It is also unexpectedly found in the invention that a pharmaceuticalcomposition comprising a combination of Valproic Acid (VA) andHydralazine (HZ) providing synergistic cancer cell-killing effects andprolonged overall survival. It was evidenced in the example that thecombination of VA and HZ-modulated chemotherapy with less than the usualchemotherapy shows an encouraging effective survival benefit in advancedHCC with manageable toxicities.

Accordingly, the present invention also provides a method for treating asubject suffering a cancer, optionally treated with a chemotherapy,comprising administering a subject in need thereof with a combination ofValproic Acid (VA) and Hydralazine (HZ) at the ratio to providesynergistic cancer cell-killing effects and prolonged overall survival.

The present invention provides a method for treating a cancer andprolonging the survival of a subject suffering a cancer treated with achemotherapy, comprising administering a subject in need thereof with acombination of valproic acid (VA) and hydrazaline (HZ) at the ratio toprovide synergistic cancer cell-killing effects and prolonged overallsurvival.

In one example of the invention, the cancer is hepatocellular carcinoma(HCC).

The pharmaceutical composition of the invention may be administered inany route that is appropriate, including but not limited to parenteralor oral administration. The pharmaceutical compositions for parenteraladministration include solutions, suspensions, emulsions, and solidinjectable compositions that are dissolved or suspended in a solventimmediately before use. The injections may be prepared by dissolving,suspending or emulsifying one or more of the active ingredients in adiluent. Examples of said diluents are distilled water for injection,physiological saline, vegetable oil, alcohol, and a combination thereof.Further, the injections may contain stabilizers, solubilizers,suspending agents, emulsifiers, soothing agents, buffers, preservatives,etc. The injections are sterilized in the final formulation step orprepared by sterile procedure.

According to the invention, the composition may be administered throughoral route, wherein the composition may be in a solid or liquid form.The solid compositions include tablets, pills, capsules, dispersiblepowders, granules, and the like. The oral compositions also includegargles which are to be stuck to oral cavity and sublingual tablets. Thecapsules include hard capsules and soft capsules. In such solidcompositions for oral use, one or more of the active compound(s) may beadmixed solely or with diluents, binders, disintegrators, lubricants,stabilizers, solubilizers, and then formulated into a preparation in aconventional manner. When necessary, such preparations may be coatedwith a coating agent, or they may be coated with two or more coatinglayers. On the other hand, the liquid compositions for oraladministration include pharmaceutically acceptable aqueous solutions,suspensions, emulsions, syrups, elixirs, and the like. In suchcompositions, one or more of the active compound(s) may be dissolved,suspended or emulsified in a commonly used diluent (such as purifiedwater, ethanol or a mixture thereof, etc.). Besides such diluents, saidcompositions may also contain wetting agents, suspending agents,emulsifiers, sweetening agents, flavoring agents, perfumes,preservatives and buffers and the like.

The present invention is further illustrated by the following examples,which are provided for the purpose of demonstration rather thanlimitation.

EXAMPLE 1

Patients

The protocol of the clinical study was reviewed and approved by theinstitute research board of Taipei Veterans General Hospital, Taipei,Taiwan. Patients aged more than 20 years with unresectable or metastaticHCC proven by pathology were recruited with an informed consent.

A Simon 2-stage phase II trial was designed with Type I error rate of0.05 and 80% power. The primary end was overall survival (OS) ofpatients with advanced hepatocellular carcinoma (HCC). The treatmentprotocol was valproic acid (VA) and hydrazaline (HZ)-modulatedgemcitabine and cisplatin as the 1^(st)-step chemotherapy, followed byVA and HZ-modulated doxorubicin and dacarbazine as the 2^(nd)-stepchemotherapy. The second ends of this trial were response rates,progression-free survivals (PFSs), and toxicities. In thirty-onepatients, eleven patients failed or were intolerant to previoussorafenib. All patients received GCGG but only 26 could receive DoxDTICtreatment. Eligibility also included Child Pugh A/B, performance status≤2; being chemonaive but regardless of previous sorafenib; having failedor being not suitable for local therapies; ECOG performance status ≤2;having adequate renal, hematological function, as indicated by aplatelet count of >75×10⁹/L, hemoglobulin >85 g/L.

Patients were treated with oral VA 200 mg thrice per day (twice for bodyweight less than 50 kg) and HZ 12.5 mg twice per day, during all thetrial days. For the 1^(st)-step chemotherapy (GCGG), patients weretreated with gemcitabine 800 mg/m² on D1, 8, and 15; cisplatin 70 mg/m²,adjusted with renal function on D1. After disease progression, ifpatients were still met the initial eligibility, they entered the2^(nd)-step chemotherapy (DoxDTIC) with intravenous doxorubicin 45 mg/m²with dacarbazine 450 mg/m² on D1 in a 21-day cycle.

Statistical Evaluations

Based on a phase III randomised trial on sorafenib in patients in theAsia-Pacific region with advanced HCC, the median overall survival was6.5 months in patient treated with sorafenib (Cheng A L, Kang Y K, ChenZ, Tsao C J, Qin S, Kim J S, Luo R, Feng J, Ye S, Yang T S, Xu J, Sun Y,Liang H, Liu J, Wang J, Tak W Y, Pan H, Burock K, Zou J, Voliotis D,Guan Z. Efficacy and safety of sorafenib in patients in the Asia-Pacificregion with advanced hepatocellular carcinoma: a phase III randomised,double-blind, placebo-controlled trial. Lancet Oncol. 2009 January;10(1):25-34). In this study, Simon's minimax two-stage design (Simon R.Optimal two-stage designs for phase II clinical trials. 1989 ControlledClinical Trials 10:1-10) was used to evaluate the effectiveness of theprotocol. Software by Dr. Ivanova from University of North Carolina atCapel Hill was used in the design. The null hypothesis that the 50% ofpatients survived longer than 6.5 months would be tested against aone-sided type I error of 0.05 and power of 0.8 when actually 70%patients survived longer than 6.5 months. In the first stage, if it isfewer than 13 out of 23 patients survived over 6.5 months, the studywould be stopped. Otherwise, 14 additional patients will be accrued fora total of 37. If more than 23 out of 37 patients survived longer than6.5 months, this protocol is assumed to be effective.

Results

Thirty-one patients were enrolled. The patients' characteristics weregiven in Table 1. All patients received GCGG but only 26 could receiveDoxDTIC treatment.

After treatment, the response rates of the patients by the ResponseEvaluation Criteria in solid tumors were given in Table 2, and therelated adverse effects caused by chemotherapy in the patients weregiven in Table 3. The progression free survivals (PFSs) of the patientstreated with VA and HZ modulated GCGG (A), DoxDTIC (B), and the 2-steptreatments (C), and overall survival of the patients with advanced HCCfollowing the VA and HZ modulated 2-step chemotherapy (D) are shown inFIG. 1.

TABLE 1 Patient's Characteristics Median age, years (range) 57 (38-77)Male, n (%) 23 (74.1) ECOG PS, n (%) 0 9 (29.0) 1 17 (54.8) 2 5 (16.1)Macroscopic vascular invasion, n (%) yes 12 (38.7) no 19 (61.3)Extrahepatic spread, n (%) 29 (93.5) Sites, n (%) lung 17 (54.8) lymphnode 9 (29.0) brain 2 (6.4) Bone and/or spine 4 (12.8) Stomach and/orintestine 3 (9.6) diaphragm 3 (9.6) Hepatic vein and/or IVC 2 (6.4)Peritoneal carcinomatosis 3 (9.6) Right ventricle 1 (3.2) Hepatitisvirus infection HBV 22 (70) HCV 2 (6.4) HBV + HCV 1 (3.2) None 6 (19.3)BCLC stage C, n (%) C 29 (93.5) B 2 (6.4) Child-Pugh score 5 13 (41.9) 611 (35.4) 7 6 (19.3) 8 1 (3.2) Max diameter of intrahepatic HCCs (cm)1.5-3   4 (12.9) 3.1-5   9 (29.0) 5.1-7.5 8 (25.8) 7.6-10  7 (22.5) >103 (9.6) Previous treatment Liver resection 12 (38.7) Livertransplantation 3 (9.6) TACE 23 (74.2) Surafenib 11 (35.4)

TABLE 2 Response rates by Response Evaluation Criteria in Solid TumorsVA + HZ/GCGG VA + HZ/DoxDTIC ( n = 31) ( n = 26) Complete response 0 (0)0 (0) Partial response 8 (25.8) 6 (23.0) Stable response 17 (54.8) 13(50.0) Progression disease 6 (19.3) 7 (26.9) DCR 25 (80.6) 20 (76.9) 50%AFP reduction 10 (32.2) 11 (42.3)

TABLE 3 Chemotherapy-related adverse effects VA + HZ/GCGG VA +HZ/DoxDTIC ( n = 31) ( n = 26) All, Grade 3/4, All, Grade 3/4, n (%) n(%) n (%) n (%) Hemorrage, UG1 1 (3.2) 1 (3.2) 1 (3.8) 1 (3.8)Neutropenic fever 2 (6.4) 2 (6.4) 5 (19.2) 2 (7.6) Liver dysfunction 3(9.6) 3 (9.6) 16 (61.5) 0 (0) Neutropenia 25 (80) 17 (54.8) 24 (92) 15(57.6) Thrombocytopenia 25 (80) 8 (25.8) 15 (57.6) 7 (26.9) Anemia 30(96.7) 50.6 (25.8) 24 (92) 3 (11.5)

Survival

The median follow up time is 14.0 months (3.2 to 45 months). The medianPFSs were 3.54 months (95% CI, 2.7 to 4.3) for the 31 patients treatedwith VA/HZ-modulated GCGG treatment and 3.74 months (95% CI, 2.67-4.81)for the 26 patients treated with VA/HZ-modulated DoxDTIC. The 2-step PFSof the 31 patients was 7.98 months (95% CI, 6.20-9.77) from the start ofVA/HZ-modulated GCGG (31 patients) to disease progression either justfailing GCGG and being unable to undertake further DoxDTIC (5 patients),or failing both GCGG and the following DoxDTIC (26 patients).

In the first stage of the Simon two stage design, we recruited 23patients, 20 patients had survivals longer than 6.5 months; therefore,additional 8 patients were recruited and 27 out of the total 31 (87%)patients had survivals longer than 6.5 months. The study was then earlyterminated for the goal of 70% had been achieved. The median OS was 17.5months (95% CI, 10.0 to 25.0). Drug related toxicities includedcytopenias, neutropenic fever and liver dysfunction with neutropenia andthrombocytopenia were the dose-limiting toxicities. The response rate(RR) of VA and HZ-modulated GCGG was 8/31 (25.8%) and disease controlrate (DRR), 25/31 (80.6%), while VA and HZ-modulated DoxDTIC had RR of5/26 (23%) and disease control rate of 20/26 (76.9%). Response rate ofVA and HZ-modulated of either GCGG or DoxDTIC is 13/31 (42%).

The progression free survivals (PFSs) of the patients treated with VAand HZ are shown in FIG. 1. It was concluded that the VA andHZ-modulated chemotherapy with less than the usual chemotherapy show anencouraging effective survival benefit in advanced HCC with manageabletoxicities. It is concluded that the PFS and OS by oxaliplatin oririnotecan-containing adjuvant chemotherapy following resection orablation of metachronous CLMs were improved.

EXAMPLE 2

Materials and Methods

Drug Treatments and Antibodies

Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich, MO) stored at−20° C. at 10 mM in dimethyl sulfoxide (DMSO) and Doxorubicin (Doxo)(Sigma-Aldrich, MO) at 3.6 mM solution were used as stock solutions.SAHA at the final concentrations of 1 μM led to 50% cell death in thethree-day growth of CTV-1. Doxo at the final concentrations of 0.09 μM,1.79 μM, 1.58 μM, and 1.03 μM respectively led to 50% cell death in thethree-day growth of CTV-1, J5, PLC and Hep3B cells. Valproic acid (VPA)(Sigma-Aldrich, MO) at 10⁻² M in dimethyl sulfoxide (DMSO), was stockedat −20° C. Fresh dilution of chemicals were prepared before theexperiments. VPA at the final concentrations of 28.67, 26.67 and 38.87μM, respectively led to 50% cell death in the three-day growth of J5,PLC and Hep3B respectively.

Cell Lines

CTV-1, Molt-3, THP-1, MCR-5, J5, LPC and Hep3B cell lines were obtainedfrom the American Type Culture Collection (Rockville, Md.). CTV-1 andMolt-3 and were grown in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS); THP-1, MCR5, J5, LPC and Hep3B were grown inDulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, N.Y.)supplemented with 10% FBS. All were grown at 37° C. in a humidifiedatmosphere containing 5% CO₂.

CTV-1 was segregated with T acute lymphoblastic leukemia and THP-1, withacute myeloid leukemia, according to their expression profiles(Andersson, 2005); Molt-3 is a T acute lymphoblastic leukemia and MRC-5,normal fetal lung fibroblasts cell line (ATCC CCL 171). J5, LPC andHep3B are HCC cell lines.

Isobologram

Dose-response interactions between SAHA and Doxo, or VPA and Doxo wereevaluated using the isobologram method of Steel and Peckham (Steel,1979). The theoretical basis of the isobologram and the procedure formaking the isobologram were described elsewhere (Akutsu, 2002; Kano,1988).

According to the dose-response curves of SAHA or VPA, and Doxo, ‘anadditive envelope’ was constructed. The location of the iso-effect dosepoints related to additive envelope were used to determine theinteractive effects of in vitro drug combinations. When the data pointsof drug combination fall within the area surrounded by lines of theadditive envelope, the combination was regarded as additive. When thedata points fall to the left of the envelope, the combination wasregarded as having a synergistic effect. When the points fall to theright of the envelope, but within the square or on the line of thesquare, the combination was regarded as having a sub-additive effect,i.e. the combination was superior or equal to a single agent, but wasless than additive. When the points fell outside the square, thecombination was regarded as having a protective effect, i.e. thecombination was inferior to a single agent in cytotoxicity. Bothsub-additive and protective effects were regarded as being antagonistic.

Apoptosis Assays

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) todetect DNA fragments and was used to apoptosis assay. We used theTACS®TdT kit (R&D, MN) according to the manufacturer's protocol.

Western Blot Analyses

After treatment for the indicated time with SAHA, Dox, or both at theconcentration of IC50, cells were lysed with 2× sodium dodecyl sulfate(SDS) buffer supplemented with protease inhibitors and subjected to aSDS-polyacrylamide gel electrophoresis (PAGE).

Subcellular Localization of γH2X and CtIP Visualized by Laser ScanningConfocal Microscopy

Cells treated for the indicated time were fixed and doublyimmunofluorescence-stained for γH2X and CtIP with mouse anti-γH2A.X(1:500) (Ser139)(05-636, Merk Millipore) and rabbit anti-CtIP (1:500)(Sc-22838, Santa Cruz) antibodies, coupled with donkeyfluorescein-conjugated anti-mouse and TRITC-conjugated anti-rabbitantibodies (Jackson ImmunoResearch, West Grove, Pa.), respectively. Thecells doubly labeled were then visualized using a Leica TCS-SP2 confocalscanning microscope equipped with an acousto-optical tunable filter anda Plan-Apochromat oil-immersion objective with a 63×/1.32 numericaperture (Leica Microsystems, Bensheim, Germany). Images were acquiredwith Leica TCSNT software.

Results

1. Synergistic Cytotoxicities of Combinations of SAHA and Doxo in CTV-1and Molt-3 T-ALL Cells and in Hep3B HCC Cells.

By isobologram analysis, all data points of iso-effect dose with SAHAand Doxo combinations administered in CTV-1 and Molt-3 T-ALL cells fellin the area of supra-additivity and implied an effect of synergistictoxicities.

In HCC cells, synergistic effects of VPA in combination with Doxo wasfound in Hep3B cells, while additive effects in J5 and antagonisticeffects in PLC cells.

As shown in FIG. 2, the survival of cells was evaluated with 2-dayculture by MTT tests. For both CTV-1 and Molt-3 cells, all theiso-effect data points of combinations of Doxo and SAHA at indicatedIC50 fractions fell in the area of synergism as shown in FIG. 2(A). ForJ5 cells, the iso-effect data showed that the combination of Doxo andVPA at indicated IC50 fractions fell in the area of additivity; for PLCcells, in the area of antagonism; while for Hep3B cells, fell in thearea of synergism, see FIG. 2(B). The additive envelopes wereconstructed according to isobologram method of Steel and Peckham (Steel& Peckham. Exploitable mechanisms in combined radiotherapy-chemotherapy:the concept of additivity. Int J Radiat Oncol Biol Phys 1979; 5:85-91).

2. SAHA Sensitized CTV-1 Cells to Doxo-Induced Apoptosis.

With 15-h cultures, the apoptosis fraction was significantly increasedwith combination of SAHA and Doxo. The increased cleaved fractions ofcaspase 8 and caspase 9 became obvious 16 hrs after treatment witheither SAHA or combination of SAHA and Doxo at IC50s. As shown in FIG.3, SAHA sensitized CTV-1 cells to doxo-induced apoptosis. The apoptosiswas evidenced with the increased M2 fractions by TUNEL assay (A), andthe increased cleavage of caspase 8 and/or 9 by western blot (B).

3. SAHA Restrained CtIP from Entering Nuclei in Sensitive CTV-1 Cellsbut not in Normal Fibroblasts.

After a 12-hour treatment with SAHA, it was found that greenfluorescence (FITC)-labeled CtIP, red fluorescence (TRITC)-labeled γH2X,and the DAPI-stained nuclei were visualized under co-focal microscopy(no figure shown), indicating. DNA break in terms of revealed by γH2Xstain occurred.

4. SAHA Increased the Cell Populations in S Phase.

The cell populations of CTV-1 were analyzed with BrdU assay after12-hour treatment with doxorubicin (Doxo), suberoylanilide hydroxamicacid (SAHA), and Doxo & SAHA. As shown in FIG. 4, the cell population inG0G1, G2M, S and subG0 phases in n each plot, R1, R2, R3 and R4respectively. It was also found that SAHA decreased CTV-1 cells in G0G1cycle arrest with increase in S phase.

The cell cycle distributions of CTV-1 after 12-hour treatment withdoxorubicin (Doxo), suberoylanilide hydroxamic acid (SAHA) at IC50 orcombinations are given in Table 4.

TABLE 4 Treatment G0G1 S G2M SubG0 Control 47.4 ± 4.1   43.1 ± 4.0   6.3 ± 2.1 0.13 ± 0.03 (vehicles) SAHA 4.13 ± 1.0   78.3 ± 6.8   11.5 ±1.2 2.0 ± 0.5 Doxo 19.4 ± 2.4 * 49.0 ± 4.2 * 28.2 ± 3.5  0.1 ± 0.04SAHA + Doxo 4.72 ± 0.5 * 66.7 ± 5.5 * 25.1 ± 3.2 0.41 ± 0.12

5. SAHA Decreased in CTV-1 Cells the Expression of CHK-1 and p-CHK-1.Which Might be Induced to Increase by Doxo.

Western blots of CtIP, p-CtIP, CHK-1, p-CHK-1, Chk-2, pChk-2 in CTV-1cell treated with SAHA, Doxo, or combination of both, at IC50 for 0, 8,12, 16 hours were observed. It was found that the expression of CHK-1and p-CHK-1 in CTV-1 cells was increased with time after exposure toDoxo, while was decreased with SAHA treatment beyond 12 hours, comparingto those treated with Doxo only or before treatment (no figure shown).

6. Differential Expression of CHK-1 in J5, PLC and Hep3B HCC CellsIntrinsic CHK-1 Expression was Relatively Low in Hep3B Independentlywith Time.

Relatively low expression was noted in Hep3B cells, in which exposure toVPA and Doxo showed synergistic cytotoxicity; while relatively highexpression in J5 and PLC cells, in which VPA and Doxo showed onlyadditive and sub-additive cytotoxicities respectively. As shown in FIGS.5(A) and (B), (A), two representive western blots for CHK-1, p-CHK-1,CHK-2 and p-CHK-2 in J5, PLC, Hep3B HCC cells treated with vehicles only(Doxo:-, and VPA: -), VPA, Doxo, or both VPA and Doxo. Low intrisicCHK-1 expression (Doxo:-, and VPA: -) showed the p-CHK-1 expression wasthe relatively low in Hep3B cells as compared to in J5 and PLC cells.(B) and (C). It was indicated that CHK-1 expression was significantlylow in Hep3B cells without exposure to VPA or Doxo or exposure to bothDoxo and VPA.

Given the above, it is concluded that the synergistic cytotoxic effectsof Doxo in combination with SAHA were found in CTV-1 and Molt-3 T-ALLcells, in which the expression and phosphorylation of CHK-1 werenegatively affected by SAHA. In the SAHA-treated T-ALL cells, the repairof Doxo-induced DSB was associated with increased γH2AX. However,although SAHA increased expression of γH2AX, apoptosis was enhanced withshortened G0G1 arrest, and low expression of CtIP and also the hamperednuclear entry of CtIP. The hampered entry of CtIP into nuclei wasrevealed by immunofluorescent confocal microscopy. HDACi thussynergistically impaired the topoisomerase inhibitor II-induced DSBrepair and enhanced apoptosis by aborting the DNA repair and inducingapoptosis by hampering the nuclear entry of CtIP. Low intrinsic CHK-1expression, which was usually accompanied by attenuated CHK-1phosphorylation, may be potentially a biomarker of synergisticcytotoxicities of HDACi in combination with DSB-inducingchemotherapeutics. Extending the implication of low CHK-1 expression tosynergistic effects of the other HDACi, valproic acid (VPA), incombination with Doxo in advanced HCC treatment, as reported in 2015ASCO meeting abstract, we employed J5, PLC and Hep3B, HCC cell lines tocheck if low intrinsic CHK-1 expression still correlated withsynergistic effects of VPA and Doxo on HCC cells. We found only in Hep3Bbut not in J5 and PLC cells, VPA in combination with Doxo showedsynergistic effects evidenced by isobologram analysis for druginteraction. Further analyses, we found relatively low intrinsic CHK-1expression significantly occurred in Hep3B cells in comparison to thatin J5 and PLC cells, in which we showed no synergistic effects of VPA incombination with Doxo. Low CHK-1 expression thus could be regarded as abiomarkers to predict synergistic effects of HDACis, such as VPA andSAHA, in combination with chemotherapeutic agents which induce doublestrained DNA break.

It is believed that a person of ordinary knowledge in the art where thepresent invention belongs can utilize the present invention to itsbroadest scope based on the descriptions herein with no need of furtherillustration. Therefore, the descriptions and claims as provided shouldbe understood as of demonstrative purpose instead of limitative in anyway to the scope of the present invention.

I claim:
 1. A method for prolonging the overall survival of a subject ina cancer chemotherapy comprising: providing a sample of cancer cells ofthe subject in a cancer chemotherapy; determining the expression ofcheckpoint kinase 1 (CHK-1) of the sample, wherein the attenuation ofactivated CHK-1 is used as a biomarker; and administering to the subjectin said cancer chemotherapy, if the attenuation of activated CHK-1 isdetected in the sample, a combination of valproic acid (VA) andhydrazaline (HZ) at the ratio to provide synergistic cancer cell-killingeffects of said chemotherapy and prolonged overall survival of saidsubject, wherein the cancer chemotherapy includes the treatment of achemotherapeutic agent selected from the group consisting gemcitabine,cisplatin, doxorubicin, dacarbazine and combination thereof.
 2. Themethod of claim 1, wherein the dosage of VA is 200 mg thrice per day ortwice for said subject's body weight less than 50 kg, the dosage of HZis 12.5 mg twice per day.
 3. The method of claim 1, wherein the canceris hepatocellular carcinoma (HCC).
 4. The method of claim 1, wherein thecancer chemotherapy includes the treatment of gemcitabine and cisplatin.5. The method of claim 1, wherein the cancer chemotherapy includes thetreatment of doxorubicin and dacarbazine.
 6. The method of claim 1,wherein the cancer chemotherapy includes the first treatment ofgemcitabine and cisplatin, and the second treatment of doxorubicin anddacarbazine.